Nonenzymatic glycation of low density lipoproteins (LDL), the major carrier of serum cholesterol, is a structural modification that affects the atherogenic potential of lipoproteins, and that increases the susceptibility of diabetic subjects to atherosclerotic complications. In particular, exposure to glycated LDL leads to intracellular accumulation of cholesterol esters, compromises the physiological ability of LDL to be metabolized by the LDL receptor pathway, and promotes uptake by alternative receptors leading to morphologic transformation of macrophages into foam cells.
Nonenzymatic glycation is a condensation reaction between carbohydrate and free amino groups at the amino-terminus or epsilon amino groups of lysine residues of proteins. The reaction is initiated with attachment of the aldehyde function of acyclic glucose to a protein amino group via nucleophilic addition, forming an aldimine, also known as a Schiff base. This intermediate product subsequently undergoes an Amadori rearrangement to form a 1-amino-1deoxyfructose derivative in stable ketoamine linkage, which in turn can cyclize to a ring structure (Cohen, M.P., Diabetes and Protein Glycosylation, Springer Verlag, 1986). This bimolecular condensation of free saccharide with protein constitutes a mechanism by which proteins are subject to post-ribosomal modification without the influence of enzymatic activities. In diabetic subjects, hyperglycemia promotes increased nonenzymatic glycation of both circulating and tissue proteins, thereby not only allowing the assessment of integrated glycemic control through determination of circulating glycated proteins, but also providing insight into the pathogenetic mechanisms responsible for the chronic complications associated with diabetes.
Glycation of LDL is pathogenetically contributory to the increased incidence of cardiovascular disease associated with diabetes. Incubation of human serum lipoproteins with glucose restilts in the covalent binding of glucose to .epsilon.-amino groups of lysine residues in the apolipoproteins of LDL, VLDL (very low density lipoproteins), and HDL (high density lipoproteins); glycation of the apolipoprotein B of LDL purified from the serum of diabetic patients is increased compared with levels of LDL glycation in samples from non-diabetic subjects. Binding and degradation of glycated LDL by cultured human fibroblasts and by umbilical vein endothelial cells is diminished compared with non-glycated LDL, and the degree of reduction in degradation is greater with increasing extent of glycation. The glycation of LDL abolishes the high affinity uptake and degradation process by normal skin fibroblasts, and also results in a decreased rate of clearance in vivo. In contrast to native LDL, which inhibits the activity of B-hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase and stimulates acyl:CoA:cholesterol acyltransferase, glycated LDL does not affect these enzymes. Thus, disturbances in receptor-mediated internalization and degradation of LDL as a result of glycation also interfere with intracellular handling of cholesterol and regulation of its synthesis. This and other evidence indicates that glycation of lysine residues on apo-LDL, which is intimately involved with LDL receptor recognition, as a consequence of hyperglycemia alters LDL metabolism and contributes to atherogenesis in diabetic subjects. Further evidence comes from the finding that the internalization and degradation of LDL from patients with insulin-dependent diabetes and poor metabolic control is decreased compared with that of LDL isolated from normal subjects or from insulin-dependent diabetic patients with good metabolic control. Modification of only 2-5% of lysine residues in LDL, a modest level of glycation comparable to that observed in some diabetic individuals, is sufficient to produce demonstrable inhibition of LDL degradation by cultured fibroblasts and of the turnover of LDL injected into guinea pigs. Plasma apolipoproteins A.sub.1, A.sub.2, B, C and E all become glycated in hyperglycemic diabetic subjects.
Alteration of LDL by glycation is one of the biochemical mechanisms leading to the formation of macrophage-derived foam cells, the major histologic marker of atherosclerosis. Like certain other lipoprotein modifications such as acetylation and malondialdehyde alteration, glycation promotes clearance by subendothelial macrophages and results in intracellular deposition of lipoprotein-derived cholesterol. Modification of critical lysine residues of the apolipoprotein B protein of LDL produces internalization by the scavenger receptor of human monocyte-macrophages and the subsequent intracellular accumulation of lipoprotein-derived cholesterol ester.
The functional consequences of apo-LDL glycation and their role in the atherogenic process make it desirable to have reliable and specific methods to quantitate the amount of LDL glycation. Existing methods to measure glycated proteins include a colorimetric procedure based on reaction with thiobarbituric acid, affinity chromatography, high pressure liquid chromatography to measure furosine, and gel electrophoresis. Each of these tests has drawbacks relating to reproducibility, cost, expensive instrumentation, accuracy or other factors, and none is specific for glycated LDL as opposed to other glycated plasma proteins. Glycated albumin can be measured specifically with a monoclonal antibody that reacts with glycated epitopes residing in albumin but not in any other protein (U.S. Pat. No. 5,223,392). Tarsio (U.S. Pat. No. 4,797,473) describes monoclonal antibodies that react preferentially with glycated serum proteins. Glycated hemoglobin can be measured specifically with a monoclonal antibody that reacts with glycated epitopes residing in hemoglobin but not in any other protein (U.S. Pat. No. 5,183,739). Knowles et al (U.S. Pat. No. 4,727,036) produced antibodies for use in determining hemoglobin A.sub.1c but these antibodies do not react with glycated epitopes residing in other proteins or in hemoglobin at positions other than the N-terminus of the beta subunit. Other antibodies against glycated proteins described in the art only react if the glycated epitope has been converted to glucitol-lysine by borohydride reduction (Curtiss and Witzum, J. Clin. Invest. 72:1427, 1983; Nakayama et al., J. Immunol. Meth. 99:95, 1987, Curtiss and Witztum, Diabetes 34:452, 1985).
It would therefore be desirable to accurately and specifically quantify the amount of glycated LDL, since its measurement provides an index of risk for cardiovascular disease. It would also be desirable to have an agent that could identify glycated LDL deposited in tissues to diagnose atherosclerosis in vivo. It would further be desirable to have an agent that could prevent the internalization of glycated LDL by cells having the potential to become atherogenic foam cells.